Field of the Invention
This invention is in the field of recombinant protein expression. In particular, the invention relates to secretion fusion partners (SFPs) and techniques for screening for suitable SFPs. Optimized SFPs for accomplishing high level secretion of target polypeptides are described. The SFPs of the invention are capable of inducing hyper-secretory production of recombinant proteins.
Related Art
The recombinant expression of proteins of interest is a widely used procedure to produce large quantities of proteins for research purposes or for therapeutic and other commercial uses. A variety of recombinant expression systems are known in the art, including bacterial, yeast, and mammalian host cell systems, and many different proteins have been successfully produced in these systems. However, there are also many proteins that are not easily produced using available expression systems, resulting in little or no protein expression and secretion. Methods for improving the secretion of recombinantly expressed proteins, such as overexpression of molecular chaperones and foldase (Hackel et al., Pharm Res 23:790 (2006); Poewer and Robinson, Biotechnol Prog 23: 364 (2007); Shusta et al., Nat Biotechnol 16: 773 (1998)), over-expression of genes related to the secretory pathway ((Carla Fama et al., Biochim Biophys Acta 1773: 232 (2007); Wentz and Shusta et al., Appl Environ Microbiol 73: 1189 (1998)), engineering of the leader sequence (Clements et al., Gene 106: 267 (1991); Kjaerulff and Jensen, Biochem Biophys Res Commun 336: 974 (2005); Sagiya et al., Microbiol. Biotechnol 42: 358 (1994); Li et al., Bitechnol Prog 18: 831 (2002)) have had some success with particular proteins of interest.
Another way of increasing protein productivity is to link the protein of interest to a fusion partner. Secretory proteins used as a fusion partners, including, human serum albumin (Kang et al., Protein Expr Purif 53: 331 (2007); Huang et al., J. Pept. Sci 14: 588 (2008)), alpha-lactalbumin (WO1995027782A1), rubredoxin (WO2000039310A1), human glucagon (WO2000053777A1), cathelicidin-related peptide derived from the hagfish (WO2005019242A2), phosphoribulokinase (US6500647B1), protein disulfide isomerase (Kajino et al., Appl Environ Microbiol 66: 638 (2000), Staphylococcal Protein A (Moreno et al., Protein Expr Purif 18: 242 (2000), Hsp150 protein (Sievi et al., Biotechnol. Prog. 19: 1368 (2003), cellulose-binding domain (Ahn et al., Appl Microbiol Biotechnol. 64: 833 (2004)) and gold binding peptide (US20050106625A1) have had some success with particular proteins of interest.
In an effort to identify secreted proteins and novel signal sequences, several signal sequence trap systems have been developed. U.S. Pat. No. 6,228,590 describes a technique for screening for mammalian signal sequences by transforming reporter protein-deficient yeast with nucleic acids comprising mammalian coding sequences fused to a reporter protein and detecting cells that secrete the reporter protein. A similar system using invertase-deficient yeast and an invertase reporter protein is disclosed in EP0907727. Yeast-based signal sequence traps have been used to identify secreted proteins from human DNA (Klein et al., Proc. Natl. Acad. Sci. USA 93:7108 (1996); Jacobs et al., Gene 198:289 (1997)), mouse DNA (Gallicioti et al., J. Membrane Biol. 183:175 (2001)), zebrafish DNA (Crosier et al., Dev. Dynamics 222:637 (2001)), Arabidopsis DNA (Goo et al., Plant Mol. Biol. 41:415 (1999)), potato DNA (Surpili et al., Anais de Academia Brasileira de Ciencias 74:599 (2002)), and Candida albicans DNA (Monteoliva et al., Eukaryotic Cell 1:514 (2002)). Similar trap systems have been developed using mammalian host cells (Gallicioti et al., J. Membrane Biol. 183:175 (2001)) and bacterial host cells (Ferguson et al., Cancer Res. 65:8209 (2000). Reporter proteins that have been used in signal sequence traps include invertase (Klein et al., Proc. Natl. Acad. Sci. USA 93:7108 (1996)), alpha amylase (U.S. Pat. No. 6,228,590), acid phosphatase (PHO5) (Surpili et al., Anais de Academia Brasileira de Ciencias 74:599 (2002)), and β-lactamase Ferguson et al., Cancer Res. 65:8209 (2000).
A method for identifying translational fusion partners (TFPs) useful for secretion of a target protein is disclosed in WO 2005/068658. The method comprises (i) obtaining a plurality of host cells transformed with a variety of vectors comprising a library of nucleic acid fragments and a target protein-encoding nucleotide sequence fused with a reporter protein-encoding nucleotide sequence, wherein the host cells are deficient in the reporter protein, and (ii) identifying a TFP library from the host cells, wherein the TFP library comprises nucleic acid fragments which individually induce the secretion of the target protein.
Translational fusion partner (TFP) technology for secretory production of rarely secretable proteins in yeast was described in WO 2007/015178. In the course of TFP screening from the yeast genome, the YGR106C (Voa1p) gene was discovered. The cellular location of Voa1p protein was recently identified in the ER membrane (Ryan et al., Mol. Biol. Cell, Epub ahead of print, Sep. 17, 2008). Voa1p was proposed to be one of five V0 assembly factors for vacuolar ATPase.
There remains a need in the art for additional sequences that enhance expression of proteins, and methods for identifying such sequences.